Recent work has convincingly shown that the degree of T cell infiltration plays a critical role in the natural progression of many cancers. A landmak study found that the type, density, and location of cytotoxic T cells within tumors enabled better prediction of patient survival than histopathological methods currently used to stage cancer. However, the tumor microenvironment strongly inhibits expansion and effector functions of tumor-specific T cells. The goal of this project is to identify novel targes for therapy by pinpointing the key genetic and biochemical defects within tumor-infiltrating T cells that restrain their effector function. The identification of such therapeutic targts is of broad relevance in oncology because T cell mediated immune responses have the potential to eradicate cancers. Towards this goal, we propose a novel in vivo shRN discovery approach that enables identification of critical genes and pathways in the relevant microenvironment. Our hypothesis is that shRNAs which target critical inhibitors within dysfunctional T cells can reprogram them to undergo substantial expansion in tumors. T cells will be genetically modified with shRNA pools and then transferred into tumor-bearing mice so that enrichment of particular shRNAs within tumors can be quantified by Illumina sequencing of the shRNA cassette. This in vivo approach will also allow us to address a second related problem in oncology, the identification of combination therapies that act in a highly synergistic manner on defined cellular pathways. We will approach this issue using a lentiviral vector with two shRNA cloning sites, so that an active shRNA can be tested for synergy against a pool of shRNAs. We will determine which shRNA combinations optimize T cell activityin vivo in terms of proliferation, cytokine production and anti-tumor cytotoxic actio. These therapeutic approaches will be tested in a mouse model in which melanomas spontaneously develop based on genetic lesions found in the human disease. A central goal of this effort is to translate these discoveries into clinical application through collaboration with a clinical investigator with expertise in adoptive T cell therapy. Adoptive transfer of T cells that express 'chimeric antigen receptors' (CARs) has emerged as a promising approach because the antibody-like extracellular domain of a CARbinds with high affinity to a surface molecule on tumor cells, while the cytoplasmic domain induces T cell activation. Co-expression of shRNAs and CARs through the same lentiviral vector into T cells could greatly enhance T cell survival and expansion within tumors. This approach will be tested using human T cells in mice bearing human melanomas, as an important step towards clinical translation. PUBLIC HEALTH RELEVANCE: The goal of this project is to discover novel targets for immunotherapy of melanoma in the relevant in vivo tissue microenvironment using a novel shRNA discovery approach. Such shRNAs may be useful for enhancing the efficacy of adoptive T cell therapy for melanoma. Some of the identified gene products could also be targeted using other approaches, such as small molecules and monoclonal antibodies. Our approach may be widely applicable for the discovery of regulators of T cell function in a variety of other human diseases.